A collaboration driving discovery
A long-standing research collaboration between Euroimmun and the University of Lübeck, Germany has led to major advances in understanding anti-p200 pemphigoid, a rare autoimmune skin blistering disease. Recent publications from the collaborative team have identified laminin subunit beta 4 (laminin β4) as a key autoantigen, demonstrated the pathogenic effects of anti-laminin β4 antibodies, and introduced a standardised indirect immunofluorescence assay (IFA) for their detection.
Examining an underdiagnosed disease
Anti-p200 pemphigoid is an autoimmune bullous dermatosis (AIBD) believed to be significantly underdiagnosed or misclassified as other AIBD. Like other pemphigoid diseases, it presents with subepidermal blisters on the skin and mucous membranes. Patients with this condition produce serum autoantibodies targeting a 200 kDa protein at the dermal-epidermal junction, which was originally identified as laminin subunit gamma 1 (laminin γ1). However, the absence of pathogenic effects from anti-laminin γ1 in vivo raised doubts as to whether it was the sole or primary antigen. [1]
Identifying a new target antigen
In 2023, the research team identified laminin β4 as a novel and highly disease-specific target antigen in anti-p200 pemphigoid using immunoprecipitation, mass spectrometry and immunoblotting. Laminin β4 is a structural protein in the basement membrane zone of the skin. Reactivity to laminin β4 was found in all patients with anti-p200 pemphigoid, but in none with other AIBD or in healthy controls — highlighting the high sensitivity and specificity of anti-laminin β4 autoantibodies for anti-p200 pemphigoid. [1]
Demonstrating pathogenic effects of anti-laminin β4
Functional studies demonstrated the pathogenic potential of anti-laminin β4 IgG both in vitro and ex vivo. Firstly, anti-laminin β4 immune complexes activated human leukocytes to release high levels of tissue-damaging molecules. Next, patient-derived anti-laminin β4 autoantibodies induced splitting of human skin cryosections, mirroring effects seen with other pemphigoid autoantibodies. In contrast, anti-laminin γ1 antibodies did not elicit such effects. Together, these findings advocate laminin β4 as the main autoantigen in the disease. [2]
Clarifying “floor” autoantibodies
In IFA, salt-split skin is a key substrate for analysing autoantibodies in pemphigoid diseases. This technique involves artificially splitting human skin samples along the dermal–epidermal junction using salt solution. Anti-laminin β4 autoantibodies bind to the dermal side of salt-split skin, generating a “floor” staining pattern, in contrast to the “roof” pattern seen with epidermal-binding autoantibodies. However, other autoantibodies also create dermal-binding staining. The most important of these are anti-collagen type VII and anti-laminin 332, which occur in epidermolysis bullosa acquisita (EBA) and a subform of mucous membrane pemphigoid (anti-laminin 332 MMP), respectively. These three autoantibodies cannot be differentiated in tissue-based IFA.
Developing reliable tools for anti-laminin β4 detection
To enable reliable monospecific detection of anti-laminin β4 autoantibodies, the research team developed a recombinant-cell IFA (RC-IFA) based on innovative Euroimmun technology. The Anti-Laminin Subunit Beta 4 (LAMB4) IIFT (for research use only) utilises transfected HEK293 cells expressing laminin β4 as the detection substrate, together with control cells. Evaluation of the test with samples from patients with anti-200 pemphigoid and control sera confirmed that the assay provides highly sensitive, specific and standardised detection of anti-laminin β4 autoantibodies. [3] Building on this, a mosaic IFA was designed to facilitate differentiation of the three major dermal-binding autoantibodies. The Dermal Binder Mosaic 1 (for research use only) comprises transfected cells expressing each of laminin β4, laminin 332 and collagen type VII, together with control cells. The substrates are incubated simultaneously, enabling efficient multiparameter autoantibody testing.
Supporting future research into anti-p200 pemphigoid
These novel assays represent critical tools for advancing research into the immunopathogenesis of anti-p200 pemphigoid. Given that anti-p200 pemphigoid is the most common pemphigoid disease associated with dermal-binding autoantibodies [4, 5], understanding its molecular basis is crucial. We look forward to continuing our collaborative research with the University of Lübeck to further unravel the role of anti-laminin β4 autoantibodies and translate these discoveries into clinical benefit.
Curious to delve deeper? Read our comprehensive article on anti-laminin β4 in Clinical Laboratory International.
[1] Goletz S, Pigors M, Lari TR, et al. Laminin is a constituent of the cutaneous basement membrane zone and additional autoantigen of anti-p200 pemphigoid. JAAD Vol 90, Issue 4, 2024, Pages 790-797. doi.org/10.1016/j.jaad.2023.11.014.
[2] Pigors M, Goletz S, Wang Y, et al. Anti-Laminin β4 IgG Drives Tissue Damage in Anti-p200 Pemphigoid and Shows Interactions with Laminin α3 and γ1/2 Chains. J Invest Dermatol. 2025 Apr;145(4):821-830.e3. doi: 10.1016/j.jid.2024.08.004.
[3] Goletz S, Probst C, Komorowski K, et al. Sensitive and specific assay for the serological diagnosis of anti-p200 pemphigoid based on the recombinant laminin β4 subunit. Br J Dermatol, Vo 191, Issue 1, July 2024, Pages 140–141. org/10.1093/bjd/ljae136
[4] Lau I, Goletz S, Holtsche MM, et al. Anti-p200 pemphigoid is the most common pemphigoid disease with serum antibodies against the dermal side by indirect immunofluorescence microscopy on human salt-split skin. J Am Acad Dermatol. 2019 Nov;81(5):1195-1197. doi: 10.1016/j.jaad.2019.03.077.
[5] Rai R, Anand JB, Shanmugasekar C et al. Anti-P 200 pemphigoid – The most common floor binding subepidermal autoimmune bullous disease in a tertiary care center in south India. Indian J Dermatol Venereol Leprol. 2021 Nov-Dec;87(6):787-791. doi: 10,25259/IJDVL_79_20.
